Dopamine-DARPP32 Feedback onto cAMP Pathway
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Dopamine-DARPP32 Feedback onto cAMP Pathway

One of the ultimate frontiers for mankind is the elucidation of the function of the mind. Dopaminergic and Glutamergic are two primary neurotransmitter systems in the brain, which are crucially important for the control of the body musculature and the Dopamine-induced signaling pathways. Dopamine, a derivative of the amino acid Tyrosine, is a monoamine neurotransmitter that serves as a chemical messenger in the nervous system. It is the predominant Catecholamine neurotransmitter in the mammalian brain, where it controls functions such as locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. Neurons in the midbrain release Dopamine, which modulates cAMP (Cyclic Adenosine 3,5-Monophosphate) production by activating DRDs (Dopamine Receptors) (Ref.1). On the basis of their molecular structure and pharmacological properties, Dopamine receptors are divided into the D1 class (DRD1 and DRD5) and D2 class (DRD2, DRD3, and DRD4). These receptors work antagonistically to modulate synthesis of cAMP, which results in stimulation and increase of the intracellular concentration of Ca2+ (Calcium Ions), leading to activation of signaling pathways. When Dopamine binds to the D1 class DRDs coupled to GN-AlphaS (GN-AlphaS Complex Locus)/(G-Alpha-Olf it leads to activation of AC (Adenylyl Cyclase), and cAMP production. In contrast, when Dopamine binds to the GN-AlphaI (Guanine Nucleotide Binding Protein-Alpha Inhibiting Activity Polypeptide)-coupled D2 class DRDs, AC activity is blocked and cAMP production is reduced. The functioning of GN-AlphaS, G-Alpha-Olf and GN-AlphaI is further aided by other G-protein subunits like GN-Beta (Guanine Nucleotide-Binding Protein-Beta) and GN-Gamma (Guanine Nucleotide-Binding Protein-Gamma) that remain attached to both the class of DRDs (Ref.2).

The effective binding and release of Dopamine is stimulated by Psychostimulants (like Cocaine, Amphetamine etc), whereas, Antipsychotics (compounds given to control symptoms of Schizophrenia and other kinds of Psychosis) inhibit the action of D2 class of receptors. A panoply of downstream effectors are activated when Dopamine binds to its receptors. The major targets include the G-Proteins, ACs, cAMP, Ca2+, (Phospholipase-C), IRKC (Inwardly Rectifying K+ Current) channels, VGKC (Voltage-Gated K+ Current) channels, L-Type CaCn (Calcium Channel), NMDAR (N-Methyl D-Aspartate Receptor)/GRIN (Glutamate Receptor Ionotropic N-Methyl D-Aspartate), AMPAR (Amino-3-Hydroxy 5-Methyl-Isoxazole-Propionic Receptor)/GRIA (Glutamate Receptor Ionotropic-AMPA) and the kinase/phosphatase inhibitor DARPP32 (Dopamine-and cAMP-Regulated Phosphoprotein) (Ref.3). The major G-Proteins that regulate activation of ACs are the GN-AlphaS, G-Alpha-Olf and GN-AlphaI. Upon activation by Dopamine and Psychostimulants, these subunits are separated from GN-Beta and GN-Gamma subunits and are converted to their GTP bound states that exhibit distinctive regulatory features on the nine tmACs (Transmembrane ACs) in order to regulate intracellular cAMP levels. The GTP bound GN-AlphaS and GN-Alpha-Olf under Dopamine and D1 class receptors stimulate ACs to convert ATP to cAMP. Increased cAMP level activates PKA (cAMP-Dependent Protein Kinase). Once active PKA phoshorylates DARPP32 at Thr34 (Threonine-34) converting it into a potent inhibitor of PPtase1 (Protein Phosphatase-1), as PPtase1 inhibits the phosphorylated forms of CREB (cAMP Response Element-Binding Protein), CREM (cAMP Response Element Modulator) and ATF1 (Activating Transcription Factor-1). Phosphorylation is a crucial event in transcriptional activation by CREB, CREM and ATF1, because it allows interaction with the transcriptional co-activators CBP (CREB-Binding Protein) and p300 leading to cell survival. PKA acts synergistically with DARPP32 (Thr34) and directly activates ATF1, CREM, CREB and CBP to enhance cell survival process. Inhibition of these transcription factors by PPtase1 leads to the disruption of communication in the Dopamine system and ultimately results in a variety of neurological psychiatric disorders, including Schizophrenia, Tourette syndrome, obsessive-compulsive disorder, Parkinson’s disease, Huntington’s disease, including drug addiction. Further targeting of PKA isozymes by AKAP9 (A-Kinase Anchor Protein-9) is important for an increasing number of physiological processes such as cAMP regulation of ion channels in the nervous system. PKA also formulates activation of ion channels like VGKC, AMPAR, NMDAR and L-Type CaCn, whereas, IRKC is directly activated by cAMP. VGKC control neuronal excitability and memory. Glutamergic neurotransmitter system release Glutamate, which along with D-Serine and Glycine stimulate the action of Dopaminergic neurotransmitter system by activating AMPAR and NMDAR , an action that is counteracted by PCP (Phencyclidine) and Ketamine (Ref.4 & 5).

In neurons, activation of the postsynaptic NMDAR , AMPAR and L-Type CaCn generate Ca2+ signals, which have a lasting impact once they are decoded by the appropriate Ca2+-sensitive proteins. Ca2+ ions modulate DARPP32 action by activating Calcium-dependent proteins like Calm (Calmodulin) and Caln (Calcineurin). Calm activation facilitates Nitric Oxide production. In response to increased intracellular Ca2+, bNOS (Brain Nitric-Oxide Synthase)/nNOS (Neuronal Nitric-oxide Synthase) interacts with Calm. The Ca2+-Calm complex, binds to bNOS and induces its translocation from the plasma membrane to the cytoplasm. Dephosphorylation of bNOS by Caln initiates the production Nitric Oxide during conversion of Arginine to Citrulline. Nitric Oxide activates sGC (Soluble Guanylyl Cyclase) that converts GTP to cGMP (Guanosine 3, 5’-Cyclic Monophosphate). This in turn activates the various cGMP-regulated signaling pathways. cGMP mediated activation of PKG (cGMP-Dependent Protein Kinase) inactivates PPtase1 function through DARPP32(Thr-34) activation (Ref.6). Dephosphorylation of bNOS activates PKA and PKC (Protein Kinase-C) signaling. Excess of Ca2+ enter the endoplasmic reticulum through SERCA (Sarcoplasmic Reticulum Ca(2+)-ATPase). However Dopamine/DRD1 stimulation activate Calcyon and GN-AlphaQ that again activate PLC to generate IP3 (Inositol 1,4,5-trisphosphate) and DAG (Diacylglycerol) by cleaving PIP2 (Phosphatidylinositol 4,5-bisphosphate). DAG activates PKC. PKA or PKC then activate bNOS by phosphorylation. Attachment of IP3 to IP3R releases Ca2+ from endoplasmic reticulum. This Ca2+ influx culminates in the stimulation of nuclear CamK4 (Calcium/Calmodulin-Dependent Protein Kinase-IV) via CamKK (Calcium/Calmodulin-Dependent Protein Kinase Kinase). CamK4 increase phosphorylation of CREB that forms a stable complex with the CBP, which in turn, recruits RNA Polymerase-II for effective transcription of Dopamine-responsive genes (Ref.7 & 8).

However Dopamine-induced gene activation is controlled when Dopamine-DARPP32 forms a feedback loop on cAMP/PKA signaling cascade. Direct feedback mechanism includes inactivation of ACs by GN-AlphaI and Par-4 (Prostate Apoptosis Response Protein-4) upon stimulation by the D2 class DRDs and Dopamine. Par-4 is a modulatory component in Dopamine signaling, Par-4/D2 class DRD complex formation is necessary to maintain an inhibitory tone on Dopamine-mediated cAMP signaling generated by low Ca2+ condition. Indirect feedback includes Calm and Caln activation by Ca2+. Calm activates Caln that in turn inhibits AMPAR, NMDAR and DARPP32 (Thr-34) action. Again Ca2+ stimulate PP2B (Protein Phosphatase-2B)/CalnA1 (Calcineurin-A1), that interferes with cAMP signaling. PP2B activates CK1 (Casein Kinase-1) in turn facilitating CDK5 (Cyclin-Dependent Kinase-5) activation. CDK5 is involved in Dopaminergic signaling in the neostriatal region of the brain. The role of CDK5 in Dopamine responses occurs through phosphorylation of DARPP32 at Thr-72 and such phosphorylation at Thr-72 inhibits the action of PKA. Secondly, PP2B dephosphorylate DARPP32at Thr-34 making it available for the action of CDK5 as well as to PKA (Ref.9). Depending on its phosphorylation state, DARPP32 is converted into an inhibitor of PPtase1 when it is phosphorylated by PKA at Threonine-34 DARPP32 is converted into an inhibitor of PKA when phosphorylated at Threonine-75 by CDK5. To balance this feedback Dopamine exerts a positive signal that increases Dopamine response by reversing phosphorylation of DARPP32 at Thr-75 by PP2A (Protein Phosphatase-2A) through activation of PKA . Dopamine signaling also stimulates Calm that competes with Par-4 and inhibits Dopamine/D2 DRD signaling. The Dopamine-DARPP32 feedback mechanisms is thus necessary to maintain the physiological state of the cell and is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities in order to avert prefrontal dysfunction associated with Schizophrenia, Parkinson’s disease, Huntington’s disease, etc. DARPP32 is a bi-functional signal transduction molecule and acts as an integrator of all the signals converging onto Dopaminoceptive cells and eventually controls the response to synaptic stimuli of all the main cellular effector systems, such as ion-channels, the secretory apparatus, the cytoskeleton, and the transcription and translating machineries. For such wide range of functions many of the drugs are targeted towards Dopamine release and its receptors in order to bring out effective treatment of neurodegenerative diseases (Ref.1 & 10).